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中华关节外科杂志(电子版) ›› 2024, Vol. 18 ›› Issue (03) : 363 -371. doi: 10.3877/cma.j.issn.1674-134X.2024.03.009

综述

单细胞RNA测序技术在骨关节炎软骨中的研究应用
陈松1, 黄玲巧2, 余清卿3, 魏志鑫3, 付琰2,()   
  1. 1. 610083 成都,解放军西部战区总医院骨科;610083 成都,四川省胰腺损伤与修复重点实验室
    2. 610083 成都,解放军西部战区总医院军队伤病员管理科
    3. 610031 成都,西南交通大学医学院
  • 收稿日期:2023-12-16 出版日期:2024-06-01
  • 通信作者: 付琰
  • 基金资助:
    四川省自然科学基金面上项目(2024NSFSC0673); 中国人民解放军西部战区总医院院管课题(2021-XZYG-B07); 中央高校基本科研业务费专项资金(2682023ZTPY052)

Research and application of single-cell RNA-sequencing technology in articular cartilage of osteoarthritis

Song Chen1, Lingqiao Huang2, Qingqing Yu3, Zhixin Wei3, Yan Fu2,()   

  1. 1. Department of Orthopaedics,The General Hospital of Western Theater Command, Chengdu 610083, China;Pancreatic Injury and Repair Key Laboratory of Sichuan Province, The General Hospital of Western Theater Command, Chengdu 610083, China
    2. Department of Military Patient Management,The General Hospital of Western Theater Command, Chengdu 610083, China
    3. College of Medicine, Southwest Jiaotong University, Chengdu 610031, China
  • Received:2023-12-16 Published:2024-06-01
  • Corresponding author: Yan Fu
引用本文:

陈松, 黄玲巧, 余清卿, 魏志鑫, 付琰. 单细胞RNA测序技术在骨关节炎软骨中的研究应用[J]. 中华关节外科杂志(电子版), 2024, 18(03): 363-371.

Song Chen, Lingqiao Huang, Qingqing Yu, Zhixin Wei, Yan Fu. Research and application of single-cell RNA-sequencing technology in articular cartilage of osteoarthritis[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2024, 18(03): 363-371.

骨关节炎(OA)的病因及发病机制尚未明确,其主要病理变化是关节软骨退变、滑膜炎症、软骨下骨重塑。目前对OA机制的研究还存在很多局限性和不准确性,如忽略了单个细胞的异质性在OA发展过程的意义。传统的RNA 测序(Bulk RNA-seq)技术获取的是组织中所有细胞或一群细胞总的RNA,分析的是平均转录组表达水平,得到的是平均数据。单细胞RNA测序(scRNA-seq)技术获取的是单个细胞转录组表达水平,可以发现细胞的异质性及分析微量转录组样品。scRNA-seq技术为从分子水平理解OA的病理机制,发现潜在的治疗靶点提供新的防治策略。本文就scRNA-seq技术及其在OA软骨中的研究应用进行概述。

The etiology and pathogenesis of osteoarthritis (OA) remain unclear and the main pathological changes are cartilage degeneration, synovial inflammation and subchondral bone remodeling. Currently, research on the mechanism of OA still has many limitations and inaccuracies, such as ignoring the significance of individual cell heterogeneity in the development of OA. Bulk RNA sequencing obtains the average gene expression level of all cell populations or a group of cell populations in the tissue and average transcriptome data. Single cell RNA-seq (scRNA-seq) obtains the gene expression level of individual cell. It plays an important role in discovering cellular heterogeneity and transcriptome analysis of trace samples, and provides new strategies for understanding the pathological mechanisms of OA at the molecular level and discovering potential therapeutic targets. This article summarized the core steps of scRNA-seq technology and its research and application in articular cartilage of OA.

图1 Bulk RNA-seq(传统RNA测序)与scRNA-seq(单细胞RNA测序)比较
Figure 1 Comparison between Bulk RNA-seq (conventional RNA sequencing) and scRNA-seq (single-cell RNA sequencing)
表1 目前主要的单细胞分离技术
Table 1 Main single cell separation techniques
表2 目前主要的转录组测序技术
Table 2 Main transcriptome sequencing techniques
单细胞转录组测序技术 技术简介 优势 不足
SMART-seq50 首先将全部RNA逆转录成cDNA,然后进行PCR扩增,再将扩增后的cDNA切割成片段,用于构建测序文库 可检测mRNA 全长,且能进行转录本异构体的分析,可以提高转录本的覆盖率 灵敏度不高,制作成本高、周期长
SMART-seq26 类似SMART-seq 灵敏度更高,扩增偏倚更低 会有部分信息丢失
SUPeR-seq51 基于均聚物尾端PCR的scRNA-seq方法,使用具有固定锚点序列的随机引物代替常用的oligo-dT引物进行cDNA合成 能够在单个细胞内检测poly(A)(+)/(-)的RNA,最大程度减少对rRNA的污染,且灵敏度和基因检测能力都更高 细胞分离过程耗时,通量低
Quartz-seq52 采用抑制性 PCR 的策略使引物自杂交形成平底锅结构来降低副产物,将小片段第二链 cDNA 形成发卡结构 极大降低 PCR 副产物 易造成扩增偏倚
CEL-seq53 利用体外线性(IVT)代替PCR实现扩增。利用含有独特条形码的oligo-dT序列将单细胞RNA逆转录成合成cDNA,通过IVT扩增获得足够的cDNA 构建测序文库 降低样本间的污染和读长偏好 存在扩增偏倚,时间和成本较高
MARS-seq50, 54 首先通过FACS分选细胞,识别不同组织和疾病的独特细胞类型,再利用3′末端计数mRNA测序方法生成cDNA 操作偏倚较少,可实现cDNA扩增的多重检测,步骤简单,通量高 时间和成本较高
STRT-seq55 将分子标记与微流控技术相结合,可定量估计起始 mRNA 的表达 减少扩增偏倚,重复性和灵敏度更高,且节约时间和成本 扩增存在序列偏好,会有信息的丢失
SCRB-seq/mcSCRB-seq56 与Smart-seq 步骤大致相同,但它使用的oligo-dT引物中包含具有细胞特异性的条形码和UMI 可以降低cDNA扩增偏差,且操作简单和成本较低 灵敏度不高,会有部分信息丢失
Drop-seq/inDrop57-58 利用微流控技术,将带有UMI的oligo-dT引物和细胞一起装入微小的液滴,细胞裂解后的mRNA与引物结合,在各单管中平行进行反转录、PCR扩增,最后生成测序文库 通量更高、速度更快,且成本更低 需要微流控平台,且会有少量信息的丢失
图2 10x Genomics单细胞转录组平台工作原理
Figure 2 The working principle of 10x Genomics single-cell transcriptome platform
图3 关节软骨结构模式图
Figure 3 Structural model of articular cartilage
图4 骨关节炎发生、发展病理变化机制
Figure 4 Development and pathogenesis osteoarthritis
表3 OA软骨中细胞亚型及功能作用
Table 3 Cell subtypes in osteoarthritis and their function
表4 健康软骨中细胞亚型
Table 4 Cell subtypes in healthy cartilage
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